So far the gene therapy for cancer has been generally achieved through intratumoral injection of therapeutic vectors. However, such topical administration is not effective for patients with metastatic tumor and recent development is directed to vectors which can specifically target metastatic tumor and makes systemic administration possible.
Sindbis viral vectors which can specifically infect metastasized tumor cells in BHK hamsters and mice to induce apoptosis (Tseng, J. C., Nature Biotechnol., 22:70-77, 2004), adenovirus VAI mutants showing cytotoxicity specifically to pancreatic cancer with ras gene mutation (Cascallo M. et al., Cancer Res., 63:5544-5550, 2003) and the like have already been reported.
Adenovirus is an icosahedron shape virus having double-stranded DNA in the genome and is classified into several categories serologically. Human adenovirus type 2 (Ad2) and human adenovirus type 5 (Ad5) are generally used for human gene therapy (e.g., JPA 2002-330789). Viral nucleic acid in adenovirus is surrounded by the protein coat called capsid. On the capsid surface, there are present 12 fibers with the tail and shaft on each vertex of its icosahedral structure and the knob at the terminus. These fibers are required for adenovirus to infect cells, and adenovirus binds to a cell surface receptor via these fibers. As such cell surface receptors, coxsackie adenovirus receptor (CAR) is known. Actually it is considered that most adenoviruses utilize CAR to adhere to cells.
Adenovirus can infect many cell-types that express CAR including normal cells and has been highly ranked for its use value as a vector. In contrast, the problem is pointed out that adenovirus infects not only targeted cells (e.g., tumor cells) but also surrounding normal cells when gene transfer is performed using an adenoviral vector. On the other hand, another problem arises that CAR is poorly expressed in pancreatic cancer cells, melanomas, etc. and adenovirus hardly infect them.
Therefore, the present inventors aimed to develop a gene transfer system by cancer-targeted viral vectors, which have high selectivity to targeted cells such as pancreatic cancer cells, etc. and can transfer and express a gene with high efficiency. To date the present inventors have prepared adenoviruses that express a reporter gene such as lacZ, EGFP, etc. using a FZ33 fiber-modified Ad5 virus, which carries a Z33 motif of protein A binding to the Fc domain of antibody in the HI loop of knob. Antibodies against membrane protein molecules CD40, CD20, or the like, which are highly expressed in leukemia cells lacking CAR expression, were previously attached to leukemia cells, recombinant adenovirus Ax3CAZ3-FZ33 expressing β-galactosidase was infected to the leukemia cells, and a gene transfer efficiency was assayed in terms of β-gal reporter gene expression as a marker. As a result, the inventors have discovered that gene expression is enhanced several fold for some of the antibodies used, as compared to control. However, targeted candidate molecules which can achieve selective and effective gene transfer to other tumor cells such as pancreatic cancer cells are not yet found.
Phosphatidic acid phosphatase (PAP) is an enzyme which catalyzes the conversion of phosphatidic acid (PA) to diacylglycerol (DG). PAP2a is an isozyme of phosphatidic acid phosphatase, was first identified in porcine and mouse (Kai et al., J. Biol. Chem., 1996, 271(31), 18931-18938), and then identified also in human (Kai et al, J. Biol. Chem., 1997, 272, 24572-24578).
It is reported that PAP2a is expressed in human normal tissues (especially, prostate gland) and prostate cancer (Leung, D. W., et al., DNA Cell Biol., 1998(4):377-85; Ulrix et al., J. Biol. Chem., 1998, 273(8):4660-4665), and Leung et al. suggested that PAP2a is down-regulated by tumorigenesis in many tissues. The invention disclosed in PCT application (WO 98/46730) by Leung et al. is also based on the finding that PAP2a is a cancer suppressor gene. According to Ultix et al., the expression in human tissues is ubiquitous but the highest level of expression was demonstrated in the prostate gland (Ultix et al., supra). It is reported that PAP2a expression in the prostate cancer culture cell line by Ultix et al. is androgen-regulated (Ultix et al., supra). However, biological importance of this protein (for example, why the protein is localized on the membrane surface, what role the protein plays in the prostate gland or prostate cancer cell line, etc.) still remains to be elucidated.